PROJECT SUMMARY/ABSTRACT This MIRA application is to support studies of how RNA binding proteins regulate choices in alternative splicing and other posttranscriptional steps in mammalian gene expression. We will continue our studies of two families of regulators, the Polypyrimidine Tract Binding Proteins and the Rbfox proteins. We will examine their molecular mechanisms of action, their biological functions, and the roles of their extended regulatory programs in neuronal development and mature neuronal function. Multiple human diseases, including several neurodegenerative disorders, involve the dysfunction of RNA binding proteins and aberrant splicing regulation. To develop treatments for such disorders, we need greater understanding of both the mechanisms and the biology of alternative splicing. We will continue our studies of the nuclear and cytoplasmic Rbfox isoforms. We will apply biochemistry and genome edited cell lines to examine how the eight RNA binding proteins of the LASR complex bind with each other, and how the assembled complex interacts with nuclear Rbfox to regulate splicing. RNAseq analyses of purified complexes and genomewide iCLIP analyses will map the binding of LASR subunits relative to the known Rbfox binding sites to reveal how the RNA within the LASR complex is organized. We will follow up on recent studies of cytoplasmic Rbfox isoforms to examine how these proteins regulate the translation and stability of mRNAs encoding important synaptic proteins, such as Vamp1. We will also continue our analyses of the Rbfox intrinsically disordered region and its ability to form molecular condensates. These analyses will be extended to IDR's in the LASR subunits to examine their homotypic and heterotypic interactions, and the role of their condensation in splicing regulation. Our studies of the mechanisms and biology of splicing repression by PTBP1 and PTBP2 will be continued. We will use biochemical methods developed in earlier work and new mass spectrometry approaches to examine the assembly and architecture of exon complexes repressed by PTBP1 and understand how PTBP1 blocks productive spliceosome assembly. We will extend our investigation of the biological impact of two transitions in neuronal splicing regulation: one induced early in neuronal development when PTBP1 is replaced with PTBP2, and one occurring when PTBP2 is downregulated late in neuronal maturation. The roles of particular splicing switches within the PTBP programs will be examined using stem cell differentiation protocols, CRISPR mediated gene editing, and whole transcriptome expression and splicing analyses. Applying RNAseq to subcellular fractions, we will characterize intron retention events controlled by PTBP1 and examine the mechanisms that sequester RNAs on chromatin. Altogether these studies will yield new understanding of the intricate molecular interactions that mediate the regulation of splicing and its misregulation in human disease.